Abstract

Human trichromatic vision is based on three classes of cones: L, M, and S (long-, middle-, and short-wavelength sensitive, respectively). Individuals can have more than one M and/or more than one L pigment gene on the X chromosome along with an S pigment gene on chromosome 7. In some people the X-linked pigment gene array can include polymorphic variants that encode multiple, spectrally distinct cone photopigment subtypes. A single-cell, polymerase chain reaction approach was used to examine visual pigment gene expression in individual human cone cells and identify them as L or M. The ratio of L:M pigment gene expression was assayed in homogenized retinal tissues taken from the same eyes. Results indicate that there is a close correspondence between the cone ratio determined from counting single cells and the L:M pigment mRNA ratio estimated from homogenized pieces of retina. The results also show that the different pigment genes in one array are often expressed at very different levels, giving rise to unequal numbers of L and M cones. Expression of only one photopigment gene was detected in each cone cell. However, individual males can have more than the classically described three spectrally distinct cone types in their retinas.

(A) Strategy for PCR amplification from single cone cells. L or M cDNA fragments containing exons 3–5, exon 3, and exon 5 were amplified. The number labels of the primers correspond to those in Table 1, where the primer sequences and their specificities are described. (B) Strategy for PCR amplification from retinal patches. L and M cDNA fragments containing a portion of exon 5 were amplified for use in experiments to estimate M/L cone ratio. A two-step process amplified L-specific exon 3 fragments. The L cDNA exon 3–5 PCR product was used in a second round of PCR to amplify L-specific exon 3.

(A) Patch of human photoreceptor mosaic obtained from a male eye donor. The image is magnified 2000× under Hoffman contrast optics. Rods are packed tightly, individual cones are transparent in this view and are located in the larger spaces separated by rods. (B) Patch of human photoreceptor beginning to self-dissociate. Planks of rods and cones are seen breaking away and dispersing from the intact mosaic.

(A) Typical field of photoreceptor cells. Individual morphologically distinguishable rods (black arrow) and cones (white arrow) are visible. A 10–15 µm micropipette guided under observation is used to collect single cones. (B) Individual cone cell alongside the micropipette. The cell was expelled into a 0.5-ml microcentrifuge tube. The image is taken through the wall of the polypropylene tube and magnified 2000× under Hoffman contrast optics to verify single-cell isolation.

PhosphorImage analysis of the ratio of M:L cDNA’s in the retina of four individuals. Amplified cDNA containing exon 5 was digested with RsaI. It is apparent with the unaided eye that each individual is expressing more L cDNA than M cDNA. Both cut (CC) and uncut (UC) controls are labeled.

PhosphorImage analysis of five single cones from one individual. Amplified cDNA containing exon 5 from the X-linked pigment genes was digested with RsaI. Each lane represents the analysis of one cone cell. Three cones expressed an L pigment gene, and two cones expressed an M pigment gene.

A 35-base-pair region of exon 5 sequence from two different single cone cells from one individual. One cell contains a cDNA sequence identical to that of an L gene, and the other contains a cDNA sequence identical to that of an M gene. Nucleotide differences that distinguish an L cone from an M cone are labeled.

PhosphorImage analysis of five single L cones from donor 54. Amplified L cDNA containing exon 3 was digested with BstYI targeting the nucleotide polymorphism at position 1026. Each lane represents a single cone cell. Two different subtypes of L genes are shown to be expressed. Three of the cone cells express the cut L gene fragment, and the other two cells express the uncut L gene fragment.

a The short form L and M indicates that the primer pair amplifies a segment from both L and M genes, L indicates that it amplifies a segment from only the L gene, and S indicates that it amplifies a segment from only the S gene.

a The short form L and M indicates that the primer pair amplifies a segment from both L and M genes, L indicates that it amplifies a segment from only the L gene, and S indicates that it amplifies a segment from only the S gene.